PhoP/Q regulated genes in Salmonella typhi identification of melittin sensitive mutants

Abstract

Many of the genes (pags (phoP activated genes) and prgs (phoP repressed genes)) regulated by the PhoP and PhoQ proteins (PhoP/Q) are necessary for survival of Salmonella typhimurium in murine macrophages and pathogenesis in mice. Although a great deal is known about the S. typhimurium phoP/Q regulon, little has been done with the human specific pathogen S. typhi, prompting us to investigate S. typhi phoP/Q regulated genes. Isogenic phoP12 (null) and phoP24 (constitutive) strains were constructed in S. typhi Ty2 and S. typhimurium C5 strains. Comparison of whole cell proteins from these strains by SDS-PAGE showed differences in both the number and molecular mass of PhoP/Q regulated proteins. This suggested that S. typhi and S. typhimurium may have different PhoP/Q regulated proteins and/or that their regulation may be different. A genetic procedure was developed to isolate mutations in PhoP/Q regulated genes. This involved random MudJ transposon mutagenesis of a phoP12 mutant, creating lacZ-gene fusions, and screening for Lac+ or Lac- colonies. A mobilizable plasmid carrying the phoP24 mutant gene was conjugated into these insertion mutants. Those that changed from Lac- to Lac+ were inferred to be pag::MudJ insertions and those that changed from Lac+ to Lac- were inferred to be prg::MudJ insertions. Five mutants with PhoP/Q regulated MudJ fusions were found by this scheme. The mutations were termed pqa (PhoPQ activated) and pqr (PhoPQ repressed) to distinguish them from other PhoP/Q regulated genes. The pqa/pqr::MudJ mutations were transduced into S. typhi phoP+ and phoP24 strains by Vi-l phage transduction. Characterization of the mutants (Southern blot analysis, beta-galactosidase activity on indicator plates and in liquid cultures) strongly suggested that their MudJ insertion mutations were in five different genes. Further characterization involved determining cationic peptide sensitivity and mouse virulence. Two mutants were found to be sensitive to the antimicrobial peptide melittin.